ABSTRACT We propose to investigate the role of non-proteolytic functions of membrane-type 1 matrix metalloproteinase (MT1- MMP, MMP-14) in the control of bone formation in vivo. MT1-MMP, a cell-membrane-bound proteinase with an extracellular catalytic site and a 20-amino acid (aa) cytoplasmic tail, degrades a variety of extracellular matrix (ECM) components and plays a key role in postnatal bone formation. The genetic deficiency of MT1-MMP (MT1-MM-/-) in the mouse causes severe osteopenia, dwarfism, faciocranial dysmorphism with lack of closure of the cranial sutures (fontanelles), and generalized arthritis. In humans, mutation of MT1-MMP causes the multicentric osteolysis and arthritis disease, Winchester syndrome, which recapitulates the dramatic skeletal phenotype of the MT1-MMP-/- mouse, showing the fundamental role of MT1-MMP in postnatal bone development in humans. Because MT1-MMP is essential in ECM remodeling, it is universally accepted that the severe phenotype of MT1-MMP-/- mice results from defective collagen turnover. However, a large body of evidence shows that the cytoplasmic tail of MT1-MMP contains multiple aa residues and motifs that control intracellular signaling pathways and cell functions by proteolysis-independent mechanisms. We generated a mouse with a point mutation of the unique tyrosine in the MT1-MMP cytoplasmic domain (MT1-MMP Y573D), and found that this non-proteolytic domain controls Wnt signaling and bone homeostasis in vivo. In the MT1- MM-/- mouse both the proteolytic and non-proteolytic functions of MT1-MMP are abolished, but the phenotypic effects are universally ascribed only to the lack of proteolytic activity. However, because MT1-MMP is a bifunctional protein, to understand its roles in vivo it is necessary to study its proteolytic activity independently of its non-proteolytic functions. This can be done by mutating the conserved Glu240 (E240A) in the MT1-MMP catalytic domain, a mutation that abrogates the proteolytic activity without affecting the non-proteolytic functions of MT1-MMP. Therefore, we propose to study the roles of MT1-MMP in bone homeostasis by developing the following Specific Aim: To generate and characterize the phenotype of mice with conditional expression of MT1-MMP E240A. We will generate a genetically modified mouse in which expression of proteolytically inactive MT1-MMP E240A can be induced in a time- and tissue-specific manner. We will then analyze the bone phenotype of conditional MMP14 E240A, MT1-MM-/- and MT1-MMP Y573D mice in which the respective mutations are induced in osteoblasts at different stages of skeletal development. Osteoblasts and mesenchymal stem cells from these mice will also be characterized for Wnt signaling and differentiation into the osteogenic lineage. We expect that MT1-MMP E240A mice devoid of MT1-MMP proteolytic activity will show milder osteopenia and dwarfism than MT1-MMP-/- mice, but a bone phenotype opposite to that of MT1-MMP Y573D mice (mild osteopenia and dwarfism vs. increased bone mass, respectively). The results will elucidate the relative contribution of the proteolysis-dependent and independent functions of MT1-MMP to bone homeostasis, and unveil non-proteolytic functions thus far undetected in vivo. The knowledge derived from our study can ultimately provide important information for the design of highly innovative approaches for the treatment of osteoporosis and other bone fragility conditions.